Implementation of the CYP Index for the Design of Selective Tryptophan-2,3-dioxygenase Inhibitors.

A class of imidazoisoindole (III) heme-binding indoleamine-2,3-dioxygenase (IDO1) inhibitors were optimized via structure-based drug design into a series of tryptophan-2,3-dioxygenase (TDO)-selective inhibitors. Kynurenine pathway modulation was demonstrated in vivo, which enabled evaluation of TDO as a potential cancer immunotherapy target. As means of mitigating the risk of drug-drug interactions arising from cytochrome P450 inhibition, a novel property-based drug design parameter, herein referred to as the CYP Index, was implemented for the design of inhibitors with appreciable selectivity for TDO over CYP3A4. We anticipate the CYP Index will be a valuable design parameter for optimizing CYP inhibition of any small molecule inhibitor containing a Lewis basic motif capable of binding heme.

Identification of Small-Molecule Noncovalent Binders Utilizing SAMDI Technology.

In recent years, the ability to unambiguously identify complex mixtures of analytes with high accuracy and resolving power in a label-free format continues to expand the application of mass spectrometry (MS) in the drug discovery process. This advantage combined with improved instrumentation makes MS suitable for targets with limited alternative assays for high-throughput screening (HTS). We describe a novel screening format using Self-Assembled Monolayers and matrix-assisted laser Desorption Ionization (SAMDI) technology. SAMDI enables affinity capture of a target protein for use in a small-molecule-binding assay format. Subsequent ionization enables the inferred identification of noncovalent compound interactions. SAMDI technology overcomes shot-to-shot variability by uniformly saturating the surface with captured protein, thereby minimizing matrix crystallization "hot spots." Furthermore, the combination with high-resolution matrix-assisted laser desorption/ionization time of flight significantly reduces interference of small-molecule detection from salt, detergent, and matrix. By using a pooled library format, the SAMDI assay can significantly improve the throughput of MS-based screening irrespective of enzyme activity. Finally, we demonstrate binding affinity rank ordering from a pool of compounds that correlates with potency data from a biochemical assay.

Differential effects of divalent manganese and magnesium on the kinase activity of leucine-rich repeat kinase 2 (LRRK2).

Various mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to susceptibility for both familial and idiopathic late-onset Parkinson's disease (PD). In this study, we have demonstrated that phosphorylation of MBP and LRRKtide by the LRRK2 G2019S mutant was activated by Mn(2+) in vitro. This enhanced G2019S kinase activity was due to the combination of an increase in kinase and a decrease in ATPase activity by Mn(2+). Compared to 10 mM Mg(2+), 1 mM Mn(2+) reduced ATP K(m) for G2019S from 103 to 1.8 muM and only modestly reduced k(cat) (2.5-fold); as a result, the Mn(2+) increased its k(cat)/K(m) by 22-fold. This change in ATP K(m) was due in large part to an increase in nucleotide affinity. While Mn(2+) also increased ATP affinity and had similar effects on k(cat)/K(m) for LRRK2 WT and R1441C enzymes, it reduced their k(cat) values significantly by 13-17-fold. Consequently, the difference in the kinase activity between G2019S and other LRRK2 variants was enhanced from about 2-fold in Mg(2+) to 10-fold in Mn(2+) at saturating ATP concentrations relative to its K(m). Furthermore, while Mg(2+) yielded optimal V(max) values at Mg(2+) concentration greater than 5 mM, the optimal Mn(2+) concentration for activating LRRK2 catalysis was in the micromolar range with increasing Mn(2+) above 1 mM causing a decrease in enzyme activity. Finally, despite the large but expected differences in IC(50) tested at 100 muM ATP, the apparent K(i) values of a small set of LRRK2 ATP-competitive inhibitors were within 5-fold between Mg(2+)- and Mn(2+)-mediated reactions except AMP-CPP, an ATP analogue.

The Aurora kinase inhibitor SNS-314 shows broad therapeutic potential with chemotherapeutics and synergy with microtubule-targeted agents in a colon carcinoma model.

Aurora kinases play key roles in regulating centrosome maturation, mitotic spindle formation, and cytokinesis during cell division, and are considered promising drug targets due to their frequent overexpression in a variety of human cancers. SNS-314 is a selective and potent pan Aurora inhibitor currently in a dose escalation phase 1 clinical trial for the treatment of patients with advanced solid tumors. Here, we report the antiproliferative effects of SNS-314 in combination with common chemotherapeutics in cell culture and xenograft models. The HCT116 colorectal carcinoma cell line, with intact or depleted p53 protein levels, was treated with SNS-314 and a cytotoxic chemotherapeutic from a panel comprised of gemcitabine, 5-fluorouracil (5-FU), carboplatin, daunomycin, SN-38 (the active metabolite of irinotecan), docetaxel, and vincristine. Combinations were administered under either concurrent or sequential schedules. SNS-314 has predominantly additive effects when administered concurrently with commonly used anticancer agents. Sequential administration of SNS-314 with chemotherapeutic compounds showed additive antiproliferative effects with carboplatin, gemcitabine, 5-FU, daunomycin, and SN-38, and synergy was observed in combination with gemcitabine, docetaxel, or vincristine. The most profound antiproliferative effects were observed with sequential administration of SNS-314 followed by docetaxel or vincristine. In vivo, SNS-314 potentiated the antitumor activity of docetaxel in xenografts. Both the in vitro synergies observed between SNS-314 and agents that target the mitotic spindle and the potentiation seen with docetaxel in vivo are consistent with a mechanism of action in which Aurora inhibition bypasses the mitotic spindle assembly checkpoint and prevents cytokinesis, augmenting subsequent spindle toxin-mediated mitotic catastrophe and cell death.

Design and synthesis of 2-amino-pyrazolopyridines as Polo-like kinase 1 inhibitors.

A series of 2-amino-pyrazolopyridines was designed and synthesized as Polo-like kinase (Plk) inhibitors based on a low micromolar hit. The SAR was developed to provide compounds exhibiting low nanomolar inhibitory activity of Plk1; the phenotype of treated cells is consistent with Plk1 inhibition. A co-crystal structure of one of these compounds with zPlk1 confirms an ATP-competitive binding mode.

Design and synthesis of 2-amino-isoxazolopyridines as Polo-like kinase inhibitors.

A series of 2-amino-isoxazolopyridines was designed and synthesized as Polo-like kinase (Plk) inhibitors. Key SAR and crystallographic data are discussed. More advanced analogues inhibit Plk1 with good enzymatic activity and modest cell-based activity. Differential selectivity among the three Plk isoforms is observed.